CoA-transferase and 3-hydroxybutyryl-CoA dehydrogenase: acetoacetyl-CoA-reacting enzymes from Clostridium beijerinckii NRRL B593

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dc.contributor.authorColby, Gary D.en
dc.contributor.committeechairChen, Jiann-Shinen
dc.contributor.committeememberAnderson, Bruce M.en
dc.contributor.committeememberDean, Dennis R.en
dc.contributor.committeememberJohnson, John L.en
dc.contributor.committeememberKrieg, Noel R.en
dc.contributor.departmentAnaerobic Microbiologyen
dc.date.accessioned2014-03-14T21:14:49Zen
dc.date.adate2006-06-07en
dc.date.available2014-03-14T21:14:49Zen
dc.date.issued1993-07-05en
dc.date.rdate2006-06-07en
dc.date.sdate2006-06-07en
dc.description.abstractIn acetone/butanol-producing clostridia, the metabolic intermediate acetoacetyl-CoA can be directed toward butyrate or butanol formation by the reaction catalyzed by 3-hydroxybutyryl-CoA dehydrogenase, or toward acetone formation by the reaction catalyzed by acetoacetate:acetate/butyrate CoA-transferase. 3-Hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 or 1.1.1.157) has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beijerinckii strain NRRL B593. The identities of 34 of the 35 N-terminal amino acid residues have been determined. The enzyme exhibited a native M<sub>r</sub> of 213,000 and a subunit M<sub>r</sub> of 30,800. It is specific for the (S)-enantiomer of 3-hydroxybutyryl-CoA. Michaelis constants for NADH and acetoacetyl-CoA were 8.6 and 14 µM, respectively. The maximum velocity of the enzyme was 540 µmol/(min mg) for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as cosubstrate; however, NAD(H) appeared to be the physiological substrate. In the presence of 9.5 µM NADH, the enzyme was inhibited by acetoacetyl-CoA at concentrations as low as 20 µM, but the inhibition was relieved as the concentration of NADH was increased, suggesting a possible mechanism for modulating the energy efficiency during growth. Acetoacetate:acetate/butyrate CoA-transferase (EC 2.8.3.9) has been purified 308-fold to apparent homogeneity from the same organism. The enzyme exhibited a native M<sub>r</sub> of 89,100. The subunits of the enzyme were separated by preparative SDS-PAGE, and exhibited M, values of 28,400 and 25,200. The identities of the 34 N-terminal amino acids of the large subunit and 38 of the 39 N-terminal amino acids of the small subunit were determined. The N-terminal region of the two subunits showed significant similarity with several other CoA transferase enzymes. Michaelis constants for butyrate and acetoacetyl-CoA were 11.7 mM and 107 µM, respectively, while those for acetate and acetoacetyl-CoA were 424 mM and 118 µM, respectively. The value of k<sub>cat</sub>/K<sub>m</sub> was approximately 100 times higher with butyrate than with acetate. Implications of the properties of these two enzymes for the acetone-butanol fermentation are discussed, and a model for the induction of the enzymes responsible for solvent production is suggested.en
dc.description.degreePh. D.en
dc.format.extentxi, 111 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-06072006-124150en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-06072006-124150/en
dc.identifier.urihttp://hdl.handle.net/10919/38541en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V856_1993.C648.pdfen
dc.relation.isformatofOCLC# 28958773en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1993.C648en
dc.subject.lcshClostridium beijerinckii -- Physiologyen
dc.subject.lcshDehydrogenasesen
dc.subject.lcshTransferasesen
dc.titleCoA-transferase and 3-hydroxybutyryl-CoA dehydrogenase: acetoacetyl-CoA-reacting enzymes from Clostridium beijerinckii NRRL B593en
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineAnaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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